Mishra Sumita, Ma Junfeng, McKoy Desirae, Sasaki Masayuki, Farinelli Federica, Page Richard C, Ranek Mark J, Zachara Natasha, Kass David A
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA.
iScience. 2023 Feb 28;26(3):106294. doi: 10.1016/j.isci.2023.106294. eCollection 2023 Mar 17.
Transient receptor potential canonical type 6 (TRPC6) is a non-voltage-gated channel that principally conducts calcium. Elevated channel activation contributes to fibrosis, hypertrophy, and proteinuria, often coupled to stimulation of nuclear factor of activated T-cells (NFAT). TRPC6 is post-translationally regulated, but a role for O-linked β-N-acetyl glucosamine (O-GlcNAcylation) as elevated by diabetes, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated at Ser14, Thr70, and Thr221 in the N-terminus ankryn-4 (AR4) and linker (LH1) domains. Mutagenesis to alanine reveals T221 as a critical controller of resting TRPC6 conductance, and associated NFAT activity and pro-hypertrophic signaling. T→A mutations at sites homologous in closely related TRPC3 and TRPC7 also increases their activity. Molecular modeling predicts interactions between Thr221--GlcNAc and Ser199, Glu200, and Glu246, and combined alanine substitutions of the latter similarly elevates resting NFAT activity. Thus, O-GlcNAcylated T221 and interactions with coordinating residues is required for normal TRPC6 channel conductance and NFAT activation.
瞬时受体电位阳离子通道蛋白6型（TRPC6）是一种非电压门控通道，主要传导钙离子。通道激活增强会导致纤维化、肥大和蛋白尿，通常与活化T细胞核因子（NFAT）的激活有关。TRPC6存在翻译后调控，但糖尿病引起的O-连接β-N-乙酰葡糖胺化（O-GlcNAcylation）的作用尚不清楚。在此，我们表明TRPC6在N端锚蛋白4（AR4）和连接子（LH1）结构域的Ser14、Thr70和Thr221位点持续发生O-GlcNAc化。突变为丙氨酸后发现Thr221是静息TRPC6电导率的关键调控因子，与相关的NFAT活性和促肥大信号传导有关。在密切相关的TRPC3和TRPC7中同源位点的T→A突变也会增加它们的活性。分子模拟预测Thr221- GlcNAc与Ser199、Glu200和Glu246之间存在相互作用，对后者进行丙氨酸联合取代同样会提高静息NFAT活性。因此，正常的TRPC6通道电导率和NFAT激活需要O-GlcNAc化的Thr221以及与协调残基的相互作用。
